The goal of these studies is to determine the required biochemical events that occur between tumor promoter-receptor interaction and the activation of effectors of neoplastic transformation. Candidate second messengers include protein phosphorylation, reactive oxygen generation, and calcium mobilization. Evidence from inhibitor studies indicates that the free radical superoxide anion is an essential early mediator of neoplastic transformation by tumor-promoting phorbol esters in JB6 mouse epidermal cells. Superoxide dismutase activity is substantially decreased by 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment of promotion sensitive (P+) but not promotion resistant (P-) cells, suggesting a causal relationship between the enzyme decrease and elevation of superoxide anion, as well as a causal relationship of these events to promotion of neoplastic transformation. Extracellular calcium acts as a required signal transducer for promotion of transformation in JB6 cells. The extracellular calcium required for TPA-promoted transformation appears to enter cells via plasma membrane channels. Cation binding to cation binding proteins may be essential signal transduction events in promotion of transformation. The trivalent cation lanthanum substitutes for calcium in activating protein kinase C, but unlike calcium, it (1) promotes transformation of JB6 cells in the absence of TPA and (2) produces conformational changes in certain protein kinase C substrates. In the future this project will extend the reactive oxygen, calcium and protein kinase signal transduction studies and converge with the promotion sensitivity (pro) gene project in order to study gene expression. Identification of lanthanum-binding promotion-relevant proteins will be pursued. Tumor promoter inducible proteins and phosphoproteins will be studied in order to identify the proteins that recognize signal sequences in pro genes.